Immobilization mediated enhancement of phyllanthin and hypophyllanthin from Phyllanthus amarus


Thakur, J.S.; Agarwal, R.K.; Kharya, M.D.

Chinese Journal of Natural Medicines 10(3): 207-212

2012


<italic>Phyllanthus amarus</italic> plant is used in the traditional system of medicine as a hepatoprotective drug for which the major lignans phyllanthin and hypophyllanthin are responsible. So far, no significant work has been done on the culture of this plant. Realizing the hepatoprotective potential, the present investigation was undertaken. A cost effective process was developed for enhancing phyllanthin and hypophyllanthin utilizing the immobilization technique. HPTLC was used to compare the phyllanthin and hypophyllanthin contents in calcium alginate immobilized cells obtained from fresh grown plants and MS medium was supplemented with different abiotic elicitors, under aseptic conditions for the treatment with chitosan, copper sulphate, phenylalanine and silver nitrate solution to make the whole process commercially viable. It was revealed that silver nitrate and phenylalanine at low concentration enhances phyllanthin and hypophyllanthin yield as compared to control immobilized cell culture. The study revealed that an increase in the content of phyllanthin and hypophyllanthin was elicitor concentration dependent and silver nitrate treatment gave a maximum yield of hepatoprotective bioactives as compared to the other abiotic elicitors used.

k
Available
online
at
www.sciencedirect.com
ScienceDirect
Chinese
Journal
of
Natural
Medicines
2012,10(3):
0207-0212
doi:
10.3724/SP.J.1009.2012.00207
Chinese
Journal
of
Natural
Medicines
ELSEVIER
Immobilization
mediated
enhancement
of
phyllanthin
and
hypophyllanthin
from
Phyllanthus
amarus
J.
S.
Thakur*,
R.
K.
Agarwal,
M.
D.
Kharya
Plant
Biotechnology
Laboratory,
Department
of
Pharmaceutical
Sciences,
Dr.
Hari
Singh
Gour
Central
University,
Sagar
(MP)
470003,
India
Available
online
May
2012
[ABSTRACT]
Phyllanthus
amarus
plant
is
used
in
the
traditional
system
of
medicine
as
a
hepatoprotective
drug
for
which
the
major
lignans
phyllanthin
and
hypophyllanthin
are
responsible.
So
far,
no
significant
work
has
been
done
on
the
culture
of
this
plant.
Realiz-
ing
the
hepatoprotective
potential,
the
present
investigation
was
undertaken.
A
cost
effective
process
was
developed
for
enhancing
phyllanthin
and
hypophyllanthin
utilizing
the
immobilization
technique.
HPTLC
was
used
to
compare
the
phyllanthin
and
hypophyl-
lanthin
contents
in
calcium
alginate
immobilized
cells
obtained
from
fresh
grown
plants
and
MS
medium
was
supplemented
with
different
abiotic
elicitors,
under
aseptic
conditions
for
the
treatment
with
chitosan,
copper
sulphate,
phenylalanine
and
silver
nitrate
solution
to
make
the
whole
process
commercially
viable.
It
was
revealed
that
silver
nitrate
and
phenylalanine
at
low
concentration
enhances
phyllanthin
and
hypophyllanthin
yield
as
compared
to
control
immobilized
cell
culture.
The
study
revealed
that
an
increase
in
the
content
of
phyllanthin
and
hypophyllanthin
was
elicitor
concentration
dependent
and
silver
nitrate
treatment
gave
a
maximum
yield
of
hepatoprotective
bioactives
as
compared
to
the
other
abiotic
elicitors
used.
[KEY
WORDS]
Immobilization;
Elicitors;
Lignans;
Hepatoprotective;
HPTLC
[CLC
Number]
R917
[Document
code]
A
[Article
ID]
1672-3651(2012)03-0207-06
1
Introduction
Biotechnology
is
an
applied
science
in
which
living
or-
ganisms
are
exploited
for
their
byproducts
for
commercial
purposes.
It
integrates
knowledge
and
information
from
many
fields.
Keeping
in
view
the
worldwide
demand
of
therapeuti-
cally
useful
bioactives,
large
scale
in
vitro
production
is
nec-
essary
for
the
future
production
of
herbal
drugs
and
their
bioactives
to
meet
their
future
global
requirement
[11
.
The
aim
of
plant
biotechnology
research
is
to
optimize
the
concentration
of
precursors,
abiotic
and
biotic
elicitors
with
duration
of
their
contact
for
maximum
response
to
the
production
of
targeted
secondary
metabolite
which
is
very
much
desired.
Liver,
one
of
the
biggest
body
organs,
per-
forms
many
vital
functions
including
digestion.
Another
im-
portant
role
is
to
detoxify
the
harmful
and
unwanted
sub-
stances
in
the
body.
During
this
detoxification
process,
the
[Received
on]
30-Jun.-2011
[*Corresponding
author]
J.
S.
Thakur:
Tel:
11-7582-222554,
Fax:
11-7582-265457,
E-mail:
jaiwantthakur@gmail.com
These
authors
have
no
any
conflict
of
interest
to
declare.
Copyright
©
2012,
China
Pharmaceutical
University.
Published
by
Elsevier
B.V.
All
rights
reserved.
liver
suffers
from
challenges
affecting
the
hepatic
architec-
ture
and
hepatocytes.
In
general,
the
liver
suffers
from
hepa-
totoxicity
which
damages
it.
Antihepatotoxic
herbs
restore
the
bile
flow
and
reduce
total
bilirubin,
biliverdin,
triglyc-
erides,
cholesterol
and
total
lipids
in
liver
[21
.
To
overcome
these
complications,
a
large
number
of
herbal
drugs
are
prescribed.
Few
such
drugs
are
(1)
Phyllan-
thus
niruri,
(2)
Ocimum
sanctum,
(3)
Tephrosia
purpurea,
(4)
Andrographis
paniculata,
(5)
Eclipta
alba
and
(6)
Terminalia
chebula
[31
.
They
contain
polyphenols,
tannins,
lignans
and
alkaloids
which
possess
serum
bilirubin,
reducing
the
effect
on
the
hepatic
tissues
without
any
adverse
effect.
These
con-
stituents
act
as
excellent
hepatoprotectives
in
the
treatment
of
jaundice
and
liver
cirrhosis
[41
.
Phyllanthus
amarus
is
an
important
hepatoprotective
drug
being
used
since
ancient
time.
The
hepatoprotective
activity
has
been
reported
from
phyllanthin
and
hypophyl-
lanthin
present
in
P.
amarus.
Although
it
is
highly
valuable
as
hepatoprotective
agent,
P
amarus
suffers
from
the
problem
of
short
supply
due
to
its
low
herbage,
availability
in
limited
duration
and
stringent
requirement
of
climatic
condition.
Phyllanthus
amarus
(Euphorbiaceae)
(commonly
called
Bhui
amla),
though
common
to
central
and
south
India,
is
indigenous
to
the
rain
forests
of
Amazon
and
other
tropical
J.
S.
Thakur,
et
al.
/Chinese
Journal
of
Natural
Medicines
2012,
10(3):
207-212
areas
of
the
world.
Due
to
its
hepatoprotective
property,
it
is
in
great
demand.
However,
scanty
growth,
short
life
span
(July
to
October)
and
requirement
of
damp
weather
for
growth
are
the
factors
for
the
short
supply
of
P
amarus.
These
factors
make
this
plant
a
suitable
candidature
for
ex-
ploitation
through
biotechnology
for
the production
of
its
hepatoprotective
bioactives
[51
.
To
overcome
these
problems,
biotechnology
has
been
used
to
produce
the
bioactives
-
phyllanthin
and
hypophyl-
lanthin
using
plant
tissue
culture
technology
utilizing
Phyl-
lanthus
amarus.
To
meet
the
increasing
demand
of
plant
based
drugs,
immobilization
of
plant
cells
is
a
viable
alternative
method
for
the production
of
therapeutic
bioactives
and
enhancing
their
yield
through
the
treatment
with
different
abiotic
and
biotic
elicitors,
precursors
and
phytohormones.
Review
of
literature
reveals
that
although
large
numbers
of
plants
possess
hepato-
protective
activity,
the
Phyllanthus
amarus
has
proved
its
hepatoprotective
potential
[61
.
Enhancement
of
secondary
metabolites
i.e.
phyllanthin
and
hypophyllanthin
in
P
amarus
plant
cells
in
vitro
was
done
by
media
manipulation,
phytohormone
regulation,
pre-
cursor
feeding
in
immobilized
and
suspension
cultures.
MS
me-
dium
was
selected
for
the
establishment
of
its
in
vitro
immobilized
cell
cultures
utilizing
R
amarus
leaves,
under
aseptic
conditions.
2
Materials
and
Methods
Fresh
leaves
of
P.
amarus
collected
from
the
medicinal
plant
garden
of
Department
of
Pharmaceutical
Sciences,
Dr.
H.
S.
Gour
Central
University,
Sagar
(M.P.)
India,
were
au-
thenticated
(Herbarium
No.QDS/3/
99/09)
from
Central
In-
stitute
of
Medicinal
and
Aromatic
Plants
(CIMAP),
Lucknow
(U.P.)
India.
From
literature
it
was
revealed
that
chitosan
and
phenylalanine
play
an
important
role
in
enhancing
the
secondary
metabolite
production
in
medicinal
plants.
It
was
also
observed
that
copper
sulphate
and
silver
nitrate
also
exert
a
noticeable
role
in
the
enhancement
of
bioac-
tives
by
immobilization
[71
.
These
abiotic
elicitors
are
therefore
selected
for
studying
their
role
in
the
production
of
important
bioac-
tives
from
P
amarus
through
immobilized
system.
2.1
Sterilization
of
material
and
preparation
of
algi-
nate
beads
for
immobilization
MS
Media
is
plant
growth
media
used
in
laborato-
ries
for
cultivation
of
plant
cell
culture.
It
was
invented
by
plant
scientists
Toshio
Murashige
and
Folke.
K.
Skoog
during
Murashige's
search
for
a
new
plant
growth
regulator.
MS
Media
was
purchased
from
Hi
media
Laboratories
pvt.
Ltd.,
and
formulated
for
study
purpose.
(product
code
:
PT810).
Dehydrated
MS
Media
34.10
g
was
dissolved
in
1000
mL
of
sterilized
distilled
water
and
the
medium
pH
was
adjusted
to
5.6.
The
freshly
prepared
MS
medium
was
autoclaved
and
stored
in
refrigerator
for
further
use
as
per
requirement.
The
collected
fresh
leaves
(25
g)
of
P
amarus
were
washed
with
running
tap
water,
followed
by
2%
tween
solu-
tion,
rewashed
thoroughly
with
distilled
water,
and
then
ster-
ilized
with
70%
ethanol.
The
leaves
were
subsequently
sur-
face
sterilized
with
0.1%
mercuric
chloride
solution
and
were
washed
thoroughly
with
sterilized
water
in
aseptic
condition.
Then
the
leaves
were
crushed
finely
using
sterilized
pestle
mortar
to
get
cell
homogenate
of
P.
amarus
leaves
[81
.
Over-night
stored
sodium
alginate
solution
20
mL
(5%)
was
mixed
thoroughly
with
the
cell
homogenate
for
30
min
to
eliminate
air
bubbles
and
to
enhance
viscosity.
The
beads,
from
cell
homogenate
were
prepared
using
25
mL
injection
syringe
and
the
prepared
beads
were
suspended
in
(2%
W/V)
calcium
chloride
solution.
The
alginate
beads
were
then
washed
with
0.9%
sterilized
saline
solution
and
transferred
into
(100
mL)
sterilized
conical
flask
containing
50
mL
MS
medium.
These
immobilized
cell
cultures
of
P
amarus
leaves
were
used
in
experimentation
for
obtaining
maximum
yield
of
phyllanthin
and
hypophyllanthin
[91
.
2.2
E
f
fect
of
chitosan
treatment
It
is
a
linear
polysaccharide
produced
commercially
by
deacetylation
of
chitin,
from
exoskeleton
of
crabs,
shrimp
etc.
and
cell
wall
of
fungi.
Chitosan
A.R (1
g)
was
dissolved
in
100
mL
of
distilled
water
by
heating
at
60
°C
for
15
minutes
with
2
mL
of
gla-
cial
acetic
acid
to
make
1%
(W/V)
chitosan
solution.
The
pH
of
the
solution
(100
mL)
was
adjusted
to
5.5
with
1
mol•L
-1
sodium
hydroxide
solution
and
autoclaved
at
120
°C
(15
lbs
/sq.inch)
for
20
minutes
and
was
filter
sterilized
before
addi-
tion
to
the
immobilized
cell
culture.
Four
sets
(in
triplicate)
of
sterilized
conical
flasks
were
taken
with
50
mL
MS
me-
dium
and
labelled
as
AC
[control]
CT1,
CT2
and
CT3
con-
taining
25
g
alginate
beads
of
P.
amarus.
Into
CT1,
CT2
and
CT3
flasks,
5,
10
and
20
mL
of
1%
chitosan
solution
was
added
and
incubated
for
14
days
in
incubator
cum
shaker
at
25
±
2
°C
between
80
to
100
rmin
-1
.
Immobilized
cell
culture
(20
mL)
from
each
flask
was
withdrawn
and
HPTLC
analysis
was
done
for
the
content
of
phyllanthin
and
hypophyllanthin
in
control
and
treated
flasks.
From
the
experimental
data
it
was
observed
that
as
the
concentration
of
chitosan
solution
in
immobilized
cell
culture
increased
from
5
to
20
mL,
the
bioactives
phyllanthin
and
hypophyllanthin enhanced
from
0.261%
to
0.405%,
which
was
higher
than
the
control
0.120%
(without
chitosan
treat-
ment).
The
maximum
increase
in
the
yield
of
bioactives
(0.405%)
with
20
mL
of
1%
chitosan
solution
was
to
the
tune
of
238%
(Table
1,
Fig.
2).
2.3
Effect
of
copper
sulphate
treatment
MS
medium
was
supplemented
with
1%
copper
sulphate
solution
and
the
stock
solution
100
mL
(conc.
1%)
was
filtered
using
sterilized
0.2
p,m
microfilter
into
a
sterile
container.
J.
S.
Thakur,
et
al.
/Chinese
Journal
of
Natural
Medicines
2012,
10(3):
207-212
Table
1
HPTLC
analysis
of
immobilized
P.
amarus
cell
sys-
tem
for
phyllanthin
and
hypophyllanthin
content
with
chito-
san
(n
=
3,
x
f
s)
Flask
Chitosan
(1%)
mL
Phyllanthin
and
hypophyllanthin
(WIW)
%
increase
com-
pared
to
control
AC
Control
0.120
±
0.001
0
CT1
5
0.261
±
0.001
117
CT2
10
0.305
±
0.002
154
CT3
20
0.405
±
0.001
238
Table
2
HPTLC
analysis
of
immobilized
P.
amarus
cell
sys-
tem
for
phyllanthin
and
hypophyllanthin
content
with
copper
sulphate
(n=
3,
x
f
s)
Copper
soul-
Phyllanthin
and
hypo-
%
increase
corn-
Flask
phate
(1%)
phyllanthin
(WM)
pared
to
control
mL
AC
Control
0.120
±
0.001
0
Cl
2
0.305
±
0.002
154
C2
4
0.420
±
0.001
250
C3
6
0.525
±
0.002
337
C4
8
0.560
±
0.001
367
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
3
2
4
5
Fig.
1
HPTLC
chromatogram
showing
the
metabolite
pro-
duction
profile
of
immobilized
P.
amarus
cell
control
2
3
0:1
02
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
.0
Fig.
2
HPTLC
chromatogram
showing
metabolite
produc-
tion
profile
of
immobilized
P.
amarus
cells
by
medium
elicita-
tion
with
20
mL
chitosan
Following
the
process
given
earlier,
the
alginate
beads
were
prepared
from
25
g
of
fresh
leaf
cell
homogenate
of
P.
amarus
aseptically.
To
four
sets
of
flasks
(in
triplicates)
la-
belled
as
C
1
,
C2,
C3
and
C4,
50
mL
MS
medium
was
taken
in
each
sterilized
flask
and
2,
4,
6
and
8
mL
of
1%
copper
sulphate
solution
was
added
from
stock
solution
and
kept
in
incubator
cum
shaker.
After
14
days,
20
mL
immobilized
cell
samples
from
each
flask
were
collected
and
analyzed
for
bioactive
contents
using
HPTLC.
Addition
of
different
concentrations
of
copper
sulphate
solution
to
MS
media
in
immobilized
cell
system
increased
the
content
of
phyllanthin
and
hypophyllanthin
compared
to
control
(0.120%)
and
maximum
enhancement
of
0.560%
was
found
with
8
mL
which
was
nearly
4
times
the
enhancement
as
compared
to
control
(Table
2,
Fig.
3).
2.4
Effect
of
phenylalanine
treatment
Phenylalanine
was
used
as
an
elicitor
for
which
1%
stock
solution
was
prepared
and
sterilized
with
0.2
lam
filter,
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Fig.
3
PTLC
chromatogram
showing
metabolite
production
profile
of
immobilized
P.
amarus
cells
by
medium
elicitation
with
8
mL
copper
sulphate
in
a
sterile
container.
In
four
sets
(in
triplicate)
of
flasks
la-
belled
as
P1,
P2,
P3
and
P4,
50
mL
MS
medium
was
added
in
each
sterilized
flask
containing
alginate
beads
prepared
from
25
g
leaf
homogenate
of
P.
amarus
by
adding
2,
4,
6
and
8
mL
of
1%
phenylalanine
solution
in
each
flask
of
the
respec-
tive
sets.
After
14
days
of
incubation
20
mL
sample
was
withdrawn
from
each
flask
and
analyzed
for
bioactives
by
HPTLC.
When
media
supplementation
was
done
by
adding
2,
4,
6
and
8
mL
of
phenylalanine
the
content
of
phyllanthin
and
hypophyllanthin
was
found
to
be
0.560%
and
0.585%
to
0.590%
and
0.615%
in
immobilized
P.
amarus
cell
system,
showing
5
times
enhancement
in
bioactives
respectively
as
compared
to
control
(0.120%)
(Table
3,
Fig.
4).
2.5
E
f
fect
of
silver
nitrate
treatment
Supplementation
of
medium
was
done
with
1%
stock
solution
of
silver
nitrate
and
thereby
sterilized.
To
four
steril-
ized
flasks
in
triplicates,
labelled
as
51,
S2,
S3
and
S4,
each
Table
3
HPTLC
analysis
of
immobilized
P.
amarus
cell
sys-
tem
for
phyllanthin
and
hypophyllanthin
content
with
phenylalanine
(n
=
3,
x
Is)
Flask
Phenylalanine
(1%)
/mL
Phyllanthin
and
hypophyllanthin
(W/W)
%
increase
com-
pared
to
control
AC
Control
0.120
±
0.001
0
P1
2
0.560
±
0.002
367
P2
4
0.585
±
0.001
387
P3
6
0.590
±
0.002
392
P4
8
0.615
±
0.001
413
i
..
a
t
ilakah.
a
2
3
4
5
J.
S.
Thakur,
et
al.
/Chinese
Journal
of
Natural
Medicines
2012,
10(3):
207-212
4
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Fig.
4
HPTLC
chromatogram
showing
metabolite
produc-
tion
profile
of
immobilized
P.
amarus
cells
by
medium
elicita-
tion
with
8
ml
Phenylalanine
containing
50
mL
of
MS
medium
alongwith
alginate
beads
of
P.
amarus
cells,
2,
4,
6
and
8
mL
of
silver
nitrate
solution
was
added
and
after
14
days
of
incubation,
20
mL
of
sample
was
withdrawn
from
each
flask
and
analyzed
for
bioactives.
When
the
immobilized
cell
system
was
supplemented
with
silver
nitrate
solution
by
adding
2,
4,
6
and
8
mL
in
MS
medium,
the
yield
of
the
phyllanthin
and
hypophyllanthin
was
found
to
be
0.690%,
0.765%,
0.770%
and
0.905%,
re-
spectively,
indicating
maximum
enhancement
of
654%
with
8m1
silver
nitrate
solution
as
compared
to
control
0.120%
(Table
4,
Fig.
5).
Table
4
HPTLC
analysis
of
immobilized
P.
amarus
cell
sys-
tem
for
phyllanthin
and
hypophyllanthin
content
with
silver
nitrate
(ts
=
4,
x
f
s)
Flask
Silvemitrate
Phyllanthin
and
(1%)
/mL
hypophyllanthin(W/W)*
%
increase
com-
pared
to
control
AC
Control
0.120
±
0.001
0
S1
2
0.690
±
0.002
475
S2
4
0.765
±
0.001
537
S3
6
0.770
±
0.002
541
S4
8
0.905
±
0.001
654
*Readings
are
average
mean
of
4
set
of
flasks
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Fig.
5
HPTLC
chromatogram
showing
metabolite
produc-
tion
profile
of
immobilized
P.
amarus
cells
by
medium
elicita-
tion
with
8
mL
silver
nitrate
nitrate
2.6
Estimation
of
phyllanthin
and
hypophyllanthin
TLC
analysis
was
done
on
silica
gel
plates
using
a
mo-
bile
phase
consisting
of
hexane:
ethyl
acetate
(2
:
1,
V/V).
Detection
of
plates
in
the
UV
light
at
254
nm
showed
the
presence
of
phyllanthin
and
hypophyllanthin.
HPTLC
was
used
for
the
qualitative
and
quantitative
analyses
of
phyllan-
thin
and
hypophyllanthin
from
immobilized
cell
system
of
P.
amarus.
After
concentration,
the
residues
were
dissolved
in
methanol
(HPTLC grade)
for
analysis.
2.7
Murugaiyah
V
and
Chan
Kit-ham
A
method
for
determination
of
four
lignans
in
Phyllan-
thus
niruri
was
developed
by
a
simple
high
performance
liquid
chromatography
(HPTLC)
method
with
fluorescence
detection.
The
highest
amount
of
lignans
was
found
in
the
leaves
of
the
plant.
HPTLC
plays
an
important
role
in
the
identification
of
medicinal
plants
and
dietary
supplements.
The
advancement
of
instrumentation
and
methodological
concepts
has
laid
a
basis
for
reliable
qualitative
and
quantitative
results
in
HPTLC.
Remarkable
progress
has
been
made
in
the
man-
ageability
of
the
entire
planer
chromatographic
process,
par-
ticularly
in
the
control
of
chromatogram
development
and
utilization
of
images
to
obtain
and
compare
information
about
separated
samples.
This
technique
is
precise,
specific
and
accurate
for
quantification
of
bioactives.
In
the
HPTLC
analysis,
the
base
line
separation
of
the
retention
times
of
standard
phyllanthin
and
hypophyllanthin
was
found
to
be
0.28
and
0.37
minutes.
Standard
curves
of
phyllanthin
and
hypophyllanthin
were
constructed
using
sim-
ple
linear
regression
at
wavelength
of
280
nm.
The
calibra-
tion
curves
were
plotted
showing
the
peak
area
of
phyllanthin
and
hypophyllanthin,
which
were
recorded
separately
and
the
percentage
W/W
was
reported
for
separate
treatments.
For
quantification
of
phyllanthin
and
hypophyllanthin
in
P.
amarus
immobilized
cells
of
14
day
samples
from
MS
medium
in
incubator
cum
shaker
were
taken.
Stock
solutions
of
phyllanthin
and
hypophyllanthin
were
prepared
by
HPTLC
grade
methanol,
to
obtain
concentration
of
200
µg
and
cali-
bration
curves
were
plotted,
using
HPTLC-integration
by
CAMAG
TLC
evaluation
software.
HPTLC
precoated
plates
at
60
°F
254
(Merck)
and
automatic
sample
III
(CAMAG)
were
used
and
integrated
with
CATS
V4.06,
S/N
:
0511A011/Sc3
V1.14,
S/N:
041123.
HPTLC
Plates
(20
cm
x
20
cm)
were
developed
using
hexane:
ethyl
acetate
solvent
system
(2
:
1)
(Application
mode
CAMAG
Automatic
TLC
Sampler
III,
Development
mode
CAMAG
Twin
Trough
Chamber)
E
n]
.
Calibration
curves
were
plotted
showing
the
peak
height
and
distance
travelled
by
the
peak
after
calculating
the
factor
x
area
divided
by
amount
of
sample
applied.
The
percentage
(W/W)
of
phyllanthin
and
hypophyllanthin
was
calculated
in
different
treated
and
control
samples
as
per
their
concentra-
tion
and
was
reported
[121
.
In
the
chromatogram
the
first
peak
seen
was
of
chlorophyll
and
the
second
peak
of
phyllanthin,
4th
peak
of
hypophyllanthin,
while
the
other
peaks
which
are
seen
are
of
other
unidentified
lignans,
terpenes
etc.
pre-
sent
in
P.
amarus,
apart
from
phyllanthin
and
hypophyllan-
thin.
J.
S.
Thakur,
et
al.
/Chinese
Journal
of
Natural
Medicines
2012,
10(3):
207-212
3
Results
and
Discussion
After
the
immobilization
of
cell
culture
of
P
amarus
in
calcium
alginate
beads
in
MS
medium,
studies
were
carried
out
to
fmd
out
the
impact
of
supplementation
with
chitosan,
copper
sulphate,
phenylalanine
and
silver
nitrate
under
asep-
tic
conditions after
immobilization
on
the
increased
accumu-
lation
of
secondary
metabolites
to
enhance
the production
of
phyllanthin
and
hypophyllanthin
as
compared
to
control.
It
is
reported that
an
optimal
concentration
of
a
suitable
sterilizing
agent
with
ideal
exposure
period
depends
on
the
nature
of
plant
for
successful
surface
sterilization
of
the
leaves.
Earlier
reports
suggested
that
0.01%
W/V
mercuric
chloride
was
found
to
be
suitable
for
surface
sterilization
of
leaves.
MS
medium
was
modified
by
adding
chitosan,
copper
sulphate,
phenyl
alanine
and
silver
nitrate
solution,
[biotic
elici-
tors]
for
enhancing
of
P
amarus
bioactives
in
immobilized
cells.
3.1
Chitosan
treatment
Immobilized
cell
system
of
Pamarus
in
MS
medium
showed
maximum
enhancement
in
the
yield
of
phyllanthin
and
hypophyllanthin
with
20
mL
of
1%
solution
of
chitosan.
It
was
238%
when
compared
to
control
immobilized
cell
cultures
after
HPTLC
analysis
.
The
entrapment
efficiency
of
chitosan
depends
on
elicitor
specificity,
cell
line
of
elicitor
used,
presence
of
growth
regulators,
composition
of
culture
medium
and
the
environmental
conditions.
Elicitation
of
immobilized
plumbago
rosea
cells
with
chitosan
proved
highly
effective
by
using
an
extracellular
site
for
the
product
accumulation
where
plumbogin
production
was
increased
about
21
times
by
collective
use
of
immobili-
zation,
elicitation
and
two
phase
culture
[13]
.
3.2
Copper
sulphate
treatment
With
immobilized
cell
system
of
P.
amarus
in
MS.
me-
dium
when
the
addition
of
2,
4,
6
and
8
mL
of
1%
copper
sulphate
solution
was
done,
the
enhancement
in
the
content
of
phyllanthin
and
hypophyllanthin
was
0.305,
0.420,
0.525
and
0.560%
W/W,
respectively.
The
maximum
enhancement
was
367%
with
8
mL
of
copper
sulphate
solution
elicitation
when
compared
to
control
immobilized
cultures
after
HPTLC
analysis.
There
are
several
reports
indicating
enhanced
production
of
secondary
metabolites
from
suspension
cultures
of
higher
plants
with
addition
of
copper
sulphate.
It
was
reported
that
copper
ions
were
suitable
for
inducing
the
accumulation
of
high
levels
of
sesquiterpenoid
phytoalexins
in
fruit
cavities
of
Datura
stramonium;
in
cell
suspension
culture,
the
highest
levels
of
products
were
formed
in
response
to
1
mmol•L
-1
copper
ions
[14]
.
3.3
Phenylalanine
treatment
Immobilized
cell
system
of
P.
amarus
in
MS
medium
elicitation
with
1%
solution
of
phenylalanine
2,
4,
6
and
8
mL
showed
enhancement in
0.560%,
0.585%,
0.590%
and
0.615%
W/W,
respectively
in
yield
of
bioactives.
The
maxi-
mum
enhancement
of
phyllanthin
and
hypophyllanthin
was
413%
in
8
mL
of
phenylalanine
when
compared
to
control
immobilized
cultures.
DiCosmo
F
and
Misawa
M
reported
that
the
addition
of
phenylalanine
into
the
agar
medium
of
Taxus
cuspidata
cells
was
found
to
stimulate
the
biosynthesis
of
taxol.
These
re-
sults
are
in
agreement
with
the
earlier
reports
of
enhanced
production
of
secondary
metabolites
with
phenylalanine
on
the
cell
cultures
of
Capsium
annuum,
Cephaelis
ipecacuanha,
Taxas
wallichtana,
T
cuspidata
etc.
[16]
.
Ballica
et
al
re-
ported
that
tropane
alkaloids
yield
was
five
times
higher
in
Datura
stramonium
cell
cultures
supplemented
with
L-phenylalanine
than
in
the
control
cultures.
3.4
Silver
nitrate
treatment
Immobilized
cell
system
of
Pamarus
in
MS
medium
elicitation
with
1%
solution
of
silver
nitrate
2,
4,
6
and
8
mL
showed
enhancement
in
0.690%,
0.765%,
0.770%
and
0.905%
W/W,
respectively
in
the
yield
of
bioactives.
The
maximum
enhancement
of
phyllanthin
and
hypophyllanthin
was
654%
in
8
mL
of
silver
nitrate
when
compared
to
control
(0.120%)
immobilized
cultures.
4
Conclusion
Immobilized
cell
cultures
of
P
amarus
with
different
treatments
in
MS
media,
the
content
of
phyllanthin
and
hy-
pophyllanthin
enhanced
maximum
with
1%
solution
of
silver
nitrate
654%
followed
by
phenylalanine
413%,
copper
sul-
phate
367%
and
chitosan
238%
when
compared
to
control
sample
0.120%
(Table
5,
Fig.
6).
A
graph
was
plotted
to
Table
5
Comparative
enhancement
of
phyllanthin
and
hypophyllanthin
in
immobilized
cell
system
with
different
treatments
(x
f
s)
Phyllanthin
and
S.
No.
Treatment
Hypophyllanthin
(Wan
1
Control
0.120
±
0.001
0
2
Chitosan
0.405
±
0.001
238
3
Copper
sulphate
0.560
±
0.001
367
4
Phenyl
alanine
0.615
±
0.001
413
5
Silver
nitrate
0.905
±
0.001
654
I I
Control
Chitosan
Copper
Phenyl
Silver
sulphate
alanine
nitrate
Treatment
Fig.
6
Comparative
percentage
enhancement
of
phyllanthin
and
hypophyllanthin
in
immobilized
cell
system
of
I?
amarus
Percentage
en-
hancement/%
700
600
500
400
'a'
300
200
100
0
J.
S.
Thakur,
et
al.
/Chinese
Journal
of
Natural
Medicines
2012,
10(3):
207-212
show
the
percentage
increase
with
different
treatments
in
immobilized
cell
system
of
P.
amarus
and
compared
with
control
in
which
the
percentage
enhancement
of
phyllanthin
and
hypophyllanthin
was
reported.
Cell
viability
studies
were
also
done
using
fluorescence
diacetate
stain
method
for
P.
amarus
in
MS
medium
of
each
supplementation
and
elicitation,
by
UV.
florescence
micro-
scope
and
it
was
found
that
66%
cells
were
living
cells
in
the
immobilized
cell
cultures
[181
.
On
the
basis
of
the
HPTLC
analysis
of
control
as
well
as
the
treated
samples
of
P.
amarus
immobilized
cell
system
[191
,
it
was
found
that
there
was
maximum
increase
in
phyllanthin
and
hypophyllanthin
yield
using
silver
nitrate
followed
by
phenylalanine,
copper
sulphate,
chitosan,
as
elicitors
when
compared
to
control
(0.120%
).
The
study
revealed
that
the
addition
of
copper
sulphate,
phenylalanine,
chitosan
and
silver
nitrate
as
biotic
elici-
tors
in
the
MS
medium
enhanced
maximum
production
of
phyllanthin
and
hypophyllanthin
in
immobilized
P.
amarus
cells
as
compared
to
control
sample.
The
production
of
hepatoprotective
bioactives,
phyllan-
thin and
hypophyllanthin
in
P.
amarus
enhanced
by
immobi-
lization
cell
system
in
MS
medium
by
supplementing
with
different
abiotic
elicitors
reported
after
HPTLC
analysis.
Acknowledgement
The
author
is
thankful
to
Mr.
Sudhakar
Agarwal,
director
and
Dr.
Jaydeep,
Incharge
Research
and
Development,
Indian
Herbs
and
Research
Supply
Co.
Saharanpur
(U.P.)
for
their
help
in
the
analytical
work.
The
author
is
also
thankful
to
AICTE
for
providing
fellowship
under
its
QIP
program.
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